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• W2886822966 abstract "The spatiotemporal regulation of gene expression is key to many biological processes. Recent imaging approaches opened exciting perspectives for understanding the intricate mechanisms regulating RNA metabolism, from synthesis to decay. Imaging techniques allow their observation at high spatial and temporal resolution, while keeping cellular morphology and micro-environment intact. Here, we focus on approaches for imaging single RNA molecules in cells, tissues, and embryos. In fixed cells, the rapid development of smFISH multiplexing opens the way to large-scale single-molecule studies, while in live cells, gene expression can be observed in real time in its native context. We highlight the strengths and limitations of these methods, as well as future challenges. We present how they advanced our understanding of gene expression heterogeneity and bursting, as well as the spatiotemporal aspects of splicing, translation, and RNA decay. These insights yield a dynamic and stochastic view of gene expression in single cells. The spatiotemporal regulation of gene expression is key to many biological processes. Recent imaging approaches opened exciting perspectives for understanding the intricate mechanisms regulating RNA metabolism, from synthesis to decay. Imaging techniques allow their observation at high spatial and temporal resolution, while keeping cellular morphology and micro-environment intact. Here, we focus on approaches for imaging single RNA molecules in cells, tissues, and embryos. In fixed cells, the rapid development of smFISH multiplexing opens the way to large-scale single-molecule studies, while in live cells, gene expression can be observed in real time in its native context. We highlight the strengths and limitations of these methods, as well as future challenges. We present how they advanced our understanding of gene expression heterogeneity and bursting, as well as the spatiotemporal aspects of splicing, translation, and RNA decay. These insights yield a dynamic and stochastic view of gene expression in single cells. “Seeing is believing and believing is knowing and knowing beats unknowing and the unknown”—Philip Roth. Imaging biological processes has revolutionized our ability to grasp the mechanisms of life. In particular, the development of single-molecule approaches in fixed and live cells opened new avenues to understand gene expression from transcription to RNA decay. In situ hybridization (ISH) can determine where specific RNAs locate in a cell or an organism, establish where RNA processing reactions take place, and measure cell-to-cell variation in gene expression. In parallel, many techniques can now image RNA and protein in live cells, giving direct access to the temporal dimension. Imaging approaches have the unique advantage of preserving cell state, morphology, and microenvironment. They revealed the dynamic and stochastic nature of gene expression at the level of single cells, and combined with image analysis and mathematical modeling, they provided unprecedented understanding of gene expression mechanisms. In the first part of this review, we present the technical developments available to image RNA metabolism at the single-molecule level. In the second part, we summarize key recent findings in transcriptional noise and in the spatiotemporal dynamics of splicing, translation, and decay, and outline current developments and challenges. ISH was invented in 1969 (Gall and Pardue, 1969Gall J.G. Pardue M.L. Formation and detection of RNA-DNA hybrid molecules in cytological preparations.Proc. Natl. Acad. Sci. USA. 1969; 63: 378-383Crossref PubMed Google Scholar) and a major breakthrough was accomplished by the Singer lab in 1998, which reported the detection of single RNA molecules in fixed cells (Femino et al., 1998Femino A.M. Fay F.S. Fogarty K. Singer R.H. Visualization of single RNA transcripts in situ.Science. 1998; 280: 585-590Crossref PubMed Scopus (673) Google Scholar). In this technique, named single-molecule fluorescent ISH (smFISH), multiple fluorescent oligonucleotides are hybridized to a target RNA, allowing detection of single molecules as diffraction-limited spots under a wide-field microscope (Figure 1). Using many probes yields high signal-to-noise ratios because non-specific signals stem from single oligonucleotides while specific signals result from many probes. Today, most smFISH variants still use multiple oligonucleotides per RNA target, from 10 to 50 (Femino et al., 1998Femino A.M. Fay F.S. Fogarty K. Singer R.H. Visualization of single RNA transcripts in situ.Science. 1998; 280: 585-590Crossref PubMed Scopus (673) Google Scholar, Raj et al., 2008Raj A. van den Bogaard P. Rifkin S.A. van Oudenaarden A. Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes.Nat. Methods. 2008; 5: 877-879Crossref PubMed Scopus (901) Google Scholar; Figure 2A). Indirect labeling schemes were also developed in which the primary probes are non-fluorescent but carry a common extra sequence named the readout (Figures 2B–2E). This sequence is hybridized to a secondary fluorescent oligonucleotide, providing flexibility in the labels and allowing synthesis of primary probes at low cost (Chen et al., 2015Chen K.H. Boettiger A.N. Moffitt J.R. Wang S. Zhuang X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells.Science. 2015; 348: aaa6090Crossref PubMed Scopus (381) Google Scholar, Choi et al., 2010Choi H.M. Chang J.Y. Trinh A. Padilla J.E. Fraser S.E. Pierce N.A. Programmable in situ amplification for multiplexed imaging of mRNA expression.Nat. Biotechnol. 2010; 28: 1208-1212Crossref PubMed Scopus (306) Google Scholar, Sinnamon and Czaplinski, 2014Sinnamon J.R. Czaplinski K. RNA detection in situ with FISH-STICs.RNA. 2014; 20: 260-266Crossref PubMed Scopus (0) Google Scholar, Tsanov et al., 2016Tsanov N. Samacoits A. Chouaib R. Traboulsi A.M. Gostan T. Weber C. Zimmer C. Zibara K. Walter T. Peter M. et al.smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability.Nucleic Acids Res. 2016; 44: e165Crossref PubMed Scopus (52) Google Scholar, Wang et al., 2012Wang F. Flanagan J. Su N. Wang L.C. Bui S. Nielson A. Wu X. Vo H.T. Ma X.J. Luo Y. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.J. Mol. Diagn. 2012; 14: 22-29Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar; Figure 2B). A good signal is often obtained with 24 oligonucleotides and smFISH is compatible with GFP detection and immuno-staining (Fusco et al., 2003Fusco D. Accornero N. Lavoie B. Shenoy S.M. Blanchard J.M. Singer R.H. Bertrand E. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells.Curr. Biol. 2003; 13: 161-167Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar).Figure 2Detection of Single RNAs in Fixed CellsShow full caption(A) SmFISH. Top: original smFISH design with 10 oligonucleotides, each 50 bases long and labeled with 5 fluorophores (red). Bottom: more recent design with 50 oligonucleotides, each 20 bases long and labeled with a single fluorophore.(B) Indirect labeling by smiFISH. A labeled secondary probe is pre-hybridized to 24 primary probes (top). The resulting duplexes are hybridized with cellular RNAs (bottom).(C) FISH-STICs. Primary probes hybridize to cellular RNAs and are labeled with three amplifier oligonucleotides (green), each of which binds five fluorescent detector oligonucleotides.(D) Branched DNA (bDNA) smFISH. Comparable to (C), except that the primary probes are pairs of contiguous oligonucleotides and binding of both is required for hybridization with the pre-amplifier.(E) Hybridization chain reaction (HCR). Two oligonucleotides form metastable hairpins (blue and green) and self-assemble into long polymers in the presence of the readout sequence, which acts as an initiator.(F) Padlock FISH. A padlock probe (green, blue, and red) takes a circular topology upon binding to target RNAs or cDNAs, and can then be covalently closed. Amplification is achieved by a rolling circle mechanism (RCA), or recursive padlock hybridization (not shown).(G) Probe generation for multiplexed smFISH. Left: structure of the primary probes. T7: T7 promoter for in vitro transcription.(H) Multiplexed smFISH. Primary probes contain two readout sequences. These are detected by successive rounds of hybridization and they together form a code that allows identification of the bound cellular RNAs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) SmFISH. Top: original smFISH design with 10 oligonucleotides, each 50 bases long and labeled with 5 fluorophores (red). Bottom: more recent design with 50 oligonucleotides, each 20 bases long and labeled with a single fluorophore. (B) Indirect labeling by smiFISH. A labeled secondary probe is pre-hybridized to 24 primary probes (top). The resulting duplexes are hybridized with cellular RNAs (bottom). (C) FISH-STICs. Primary probes hybridize to cellular RNAs and are labeled with three amplifier oligonucleotides (green), each of which binds five fluorescent detector oligonucleotides. (D) Branched DNA (bDNA) smFISH. Comparable to (C), except that the primary probes are pairs of contiguous oligonucleotides and binding of both is required for hybridization with the pre-amplifier. (E) Hybridization chain reaction (HCR). Two oligonucleotides form metastable hairpins (blue and green) and self-assemble into long polymers in the presence of the readout sequence, which acts as an initiator. (F) Padlock FISH. A padlock probe (green, blue, and red) takes a circular topology upon binding to target RNAs or cDNAs, and can then be covalently closed. Amplification is achieved by a rolling circle mechanism (RCA), or recursive padlock hybridization (not shown). (G) Probe generation for multiplexed smFISH. Left: structure of the primary probes. T7: T7 promoter for in vitro transcription. (H) Multiplexed smFISH. Primary probes contain two readout sequences. These are detected by successive rounds of hybridization and they together form a code that allows identification of the bound cellular RNAs. In optically challenging samples, smFISH may require amplification. Colorimetric and fluorescent enzymatic methods can be used, but DNA-based amplification schemes provide better signals (Sylwestrak et al., 2016Sylwestrak E.L. Rajasethupathy P. Wright M.A. Jaffe A. Deisseroth K. Multiplexed intact-tissue transcriptional analysis at cellular resolution.Cell. 2016; 164: 792-804Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). FISH-STICs and branched DNA (bDNA) are related techniques that involve successive hybridizations of pre-amplifiers, amplifiers, and detector oligonucleotides to the readout sequence of unlabeled primary probes (Sinnamon and Czaplinski, 2014Sinnamon J.R. Czaplinski K. RNA detection in situ with FISH-STICs.RNA. 2014; 20: 260-266Crossref PubMed Scopus (0) Google Scholar, Wang et al., 2012Wang F. Flanagan J. Su N. Wang L.C. Bui S. Nielson A. Wu X. Vo H.T. Ma X.J. Luo Y. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.J. Mol. Diagn. 2012; 14: 22-29Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar; Figures 2C and 2D). Hybridization chain reaction (HCR) involves the continuous binding of two complementary hairpins on the readout and longer hybridization duration yields more amplification (Choi et al., 2010Choi H.M. Chang J.Y. Trinh A. Padilla J.E. Fraser S.E. Pierce N.A. Programmable in situ amplification for multiplexed imaging of mRNA expression.Nat. Biotechnol. 2010; 28: 1208-1212Crossref PubMed Scopus (306) Google Scholar; Figure 2E). These methods yield 10- to 100-fold signal enhancement but at the cost of more complex experimental workflows. Amplification is also not uniform across RNAs, potentially confounding RNA aggregates with bright single molecules. Another way of improving contrasts is to use clearing techniques (Long et al., 2017Long X. Colonell J. Wong A.M. Singer R.H. Lionnet T. Quantitative mRNA imaging throughout the entire Drosophila brain.Nat. Methods. 2017; 14: 703-706Crossref PubMed Scopus (29) Google Scholar, Moffitt et al., 2016Moffitt J.R. Hao J. Bambah-Mukku D. Lu T. Dulac C. Zhuang X. High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing.Proc. Natl. Acad. Sci. USA. 2016; 113: 14456-14461Crossref PubMed Scopus (1) Google Scholar, Shah et al., 2016Shah S. Lubeck E. Schwarzkopf M. He T.F. Greenbaum A. Sohn C.H. Lignell A. Choi H.M. Gradinaru V. Pierce N.A. Cai L. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.Development. 2016; 143: 2862-2867Crossref PubMed Google Scholar, Sylwestrak et al., 2016Sylwestrak E.L. Rajasethupathy P. Wright M.A. Jaffe A. Deisseroth K. Multiplexed intact-tissue transcriptional analysis at cellular resolution.Cell. 2016; 164: 792-804Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In general, these different approaches are well suited for imaging with low numerical aperture objectives, as well as in samples that are thick or with high background fluorescence such as embryos or tissues. RNAs shorter than 0.5–1 kb are difficult to visualize because they cannot bind many probes. Yet many non-coding RNAs and alternative exons are in this length range or shorter. Amplification systems optimized for specificity are well suited for short RNAs. Padlock probes can be covalently closed and amplified only when they bind to the correct target RNA (Larsson et al., 2010Larsson C. Grundberg I. Söderberg O. Nilsson M. In situ detection and genotyping of individual mRNA molecules.Nat. Methods. 2010; 7: 395-397Crossref PubMed Scopus (203) Google Scholar, Rouhanifard et al., 2017Rouhanifard S.H. Dunagin M. Mellis I.A. Bayatpour S. Symmons O. Cote A. Raj A. Single-molecule fluorescent amplification of RNA using clampFISH probes.bioRxiv. 2017; https://doi.org/10.1101/222794Crossref Google Scholar; Figure 2F). Similarly, the bDNA approach requires the binding of two contiguous pre-amplifiers for successful amplification (Wang et al., 2012Wang F. Flanagan J. Su N. Wang L.C. Bui S. Nielson A. Wu X. Vo H.T. Ma X.J. Luo Y. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.J. Mol. Diagn. 2012; 14: 22-29Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar; Figure 2D). The limitations of these approaches reside in their long protocols and in the small number of probes that lowers the detection rate; for instance, if probe binding sites are not accessible in all RNA molecules. The high amplification can also create false positives. These methods are, however, powerful and can detect even single microRNA (miRNA) molecules (Larsson et al., 2010Larsson C. Grundberg I. Söderberg O. Nilsson M. In situ detection and genotyping of individual mRNA molecules.Nat. Methods. 2010; 7: 395-397Crossref PubMed Scopus (203) Google Scholar). The first large-scale FISH study was performed in Drosophila embryos (Lécuyer et al., 2007Lécuyer E. Yoshida H. Parthasarathy N. Alm C. Babak T. Cerovina T. Hughes T.R. Tomancak P. Krause H.M. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.Cell. 2007; 131: 174-187Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). The approach did not reach single-molecule sensitivity but nevertheless led to a remarkable discovery. Indeed, 70% of the 2,300 analyzed mRNAs displayed specific sub-cellular localization patterns, thus revealing that RNA localization is widespread in this organism. In cultured cells, many RNAs are present in few copies per cell and their detection thus requires single-molecule sensitivity. The first systematic smFISH study analyzed 900 human mRNAs with bDNA amplification and low-magnification objectives, while oligonucleotide probes were synthesized individually (Battich et al., 2013Battich N. Stoeger T. Pelkmans L. Image-based transcriptomics in thousands of single human cells at single-molecule resolution.Nat. Methods. 2013; 10: 1127-1133Crossref PubMed Scopus (112) Google Scholar). A cost-effective strategy to scale up smFISH is multiplexing. Current implementations, named merFISH and seqFISH (Chen et al., 2015Chen K.H. Boettiger A.N. Moffitt J.R. Wang S. Zhuang X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells.Science. 2015; 348: aaa6090Crossref PubMed Scopus (381) Google Scholar, Lubeck et al., 2014Lubeck E. Coskun A.F. Zhiyentayev T. Ahmad M. Cai L. Single-cell in situ RNA profiling by sequential hybridization.Nat. Methods. 2014; 11: 360-361Crossref PubMed Scopus (193) Google Scholar), allow the detection of up to 10,000 RNA species in the same cell, opening the door to image-based transcriptomics (Shah et al., 2018Shah S. Takei Y. Zhou W. Lubeck E. Yun J. Eng C.L. Koulena N. Cronin C. Karp C. Liaw E.J. et al.Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH.Cell. 2018; 174: 363-376.e16Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). These methods can reveal RNA localization and nuclear organization, identify co-regulated genes, and characterize new cell types (Chen et al., 2015Chen K.H. Boettiger A.N. Moffitt J.R. Wang S. Zhuang X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells.Science. 2015; 348: aaa6090Crossref PubMed Scopus (381) Google Scholar, Shah et al., 2016Shah S. Lubeck E. Schwarzkopf M. He T.F. Greenbaum A. Sohn C.H. Lignell A. Choi H.M. Gradinaru V. Pierce N.A. Cai L. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.Development. 2016; 143: 2862-2867Crossref PubMed Google Scholar, Shah et al., 2018Shah S. Takei Y. Zhou W. Lubeck E. Yun J. Eng C.L. Koulena N. Cronin C. Karp C. Liaw E.J. et al.Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH.Cell. 2018; 174: 363-376.e16Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Probes are generated by parallel on-chip synthesis, yielding mixtures of 10,000 to 100,000 oligonucleotides. Probes are then amplified by PCR and converted to single strand by in vitro transcription and reverse transcription (Figure 2G). High levels of multiplexing require encoding schemes, in which the probes for a single RNA species carry multiple readout sequences (Figure 2H). These are detected by sequential rounds of hybridization, using either one or multiple colors at a time (Chen et al., 2015Chen K.H. Boettiger A.N. Moffitt J.R. Wang S. Zhuang X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells.Science. 2015; 348: aaa6090Crossref PubMed Scopus (381) Google Scholar, Lubeck et al., 2014Lubeck E. Coskun A.F. Zhiyentayev T. Ahmad M. Cai L. Single-cell in situ RNA profiling by sequential hybridization.Nat. Methods. 2014; 11: 360-361Crossref PubMed Scopus (193) Google Scholar, Shah et al., 2018Shah S. Takei Y. Zhou W. Lubeck E. Yun J. Eng C.L. Koulena N. Cronin C. Karp C. Liaw E.J. et al.Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH.Cell. 2018; 174: 363-376.e16Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Each readout sequence is analogous to a digital bit, and in theory, n readouts allow for 2n − 1 codes (i.e., RNA species; Figure 2H). The encoding capacity is, however, voluntarily reduced to include error correction schemes, such that the correct code can still be identified with single reading errors. Another limitation is that encoding requires all transcripts to be spatially separated, a condition no longer met if abundant or numerous transcripts are imaged. It is, however, possible to use super-resolution approaches to resolve higher densities of RNA molecules (see below). RNA molecules that are too abundant or that concentrate in a small area cannot be resolved by standard microscopy. Structured illumination provides a 2-fold improvement in resolution in each dimension and has been successfully applied to smFISH without experimental modifications (Tantale et al., 2016Tantale K. Mueller F. Kozulic-Pirher A. Lesne A. Victor J.M. Robert M.C. Capozi S. Chouaib R. Bäcker V. Mateos-Langerak J. et al.A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting.Nat. Commun. 2016; 7: 12248Crossref PubMed Google Scholar, Trcek et al., 2015Trcek T. Grosch M. York A. Shroff H. Lionnet T. Lehmann R. Drosophila germ granules are structured and contain homotypic mRNA clusters.Nat. Commun. 2015; 6: 7962Crossref PubMed Google Scholar). Interestingly, smFISH can also be combined with expansion microscopy to increase spatial resolution (Chen et al., 2016Chen F. Wassie A.T. Cote A.J. Sinha A. Alon S. Asano S. Daugharthy E.R. Chang J.B. Marblestone A. Church G.M. et al.Nanoscale imaging of RNA with expansion microscopy.Nat. Methods. 2016; 13: 679-684Crossref PubMed Scopus (86) Google Scholar, Tsanov et al., 2016Tsanov N. Samacoits A. Chouaib R. Traboulsi A.M. Gostan T. Weber C. Zimmer C. Zibara K. Walter T. Peter M. et al.smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability.Nucleic Acids Res. 2016; 44: e165Crossref PubMed Scopus (52) Google Scholar, Wang et al., 2018Wang G. Moffitt J.R. Zhuang X. Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy.Sci. Rep. 2018; 8: 4847Crossref PubMed Scopus (28) Google Scholar; Figure 1B). Here, RNAs are anchored to a swellable polymer, either non-specifically (Chen et al., 2016Chen F. Wassie A.T. Cote A.J. Sinha A. Alon S. Asano S. Daugharthy E.R. Chang J.B. Marblestone A. Church G.M. et al.Nanoscale imaging of RNA with expansion microscopy.Nat. Methods. 2016; 13: 679-684Crossref PubMed Scopus (86) Google Scholar, Wang et al., 2018Wang G. Moffitt J.R. Zhuang X. Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy.Sci. Rep. 2018; 8: 4847Crossref PubMed Scopus (28) Google Scholar) or via the readout sequence (Tsanov et al., 2016Tsanov N. Samacoits A. Chouaib R. Traboulsi A.M. Gostan T. Weber C. Zimmer C. Zibara K. Walter T. Peter M. et al.smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability.Nucleic Acids Res. 2016; 44: e165Crossref PubMed Scopus (52) Google Scholar). The polymer expands after proteolysis and addition of water, yielding a resolution increase of about 3- to 4-fold in every dimension. While the procedure is more complex, the expanded samples can be directly observed on standard wide-field microscopes, and polymer embedding also reduces background fluorescence. Multiplexed smFISH techniques are rapidly evolving and may soon allow a dramatic shift in gene expression studies. In particular, multiplexed smFISH is now an appealing alternative to single-cell sequencing (see Chen et al., 2018Chen X. Teichmann S. Meyer K. From tissues to cell types and back: single-cell gene expression analysis of tissues architecture.Annu. Rev. Biomed. Data Sci. 2018; 1: 29-51Crossref Google Scholar for a comparison of these techniques), with the advantage of having a direct RNA detection method ideally suited for low-abundance RNAs, while providing information on cell shape, micro-environment, and sub-cellular localization. Designing efficient probes is a key step in smFISH. A number of algorithms are available that mostly homogenize the Tm or ΔG° of the probes. However, other factors impact hybridization, including sequence features, base composition, and secondary structure of the probe and target RNAs. These could be included in next-generation design algorithms. High-throughput and multiplexed datasets provide very rich spatial information, and appropriate image analysis tools are essential to help visualize and interpret these data. First, cells and individual RNA molecules have to be robustly and automatically detected, ideally in 3D. Suitable approaches have been developed for cell lines and multicellular organisms such as Drosophila and zebrafish and can be further improved (Mueller et al., 2013Mueller F. Senecal A. Tantale K. Marie-Nelly H. Ly N. Collin O. Basyuk E. Bertrand E. Darzacq X. Zimmer C. FISH-quant: automatic counting of transcripts in 3D FISH images.Nat. Methods. 2013; 10: 277-278Crossref PubMed Scopus (108) Google Scholar, Stapel et al., 2016Stapel L.C. Lombardot B. Broaddus C. Kainmueller D. Jug F. Myers E.W. Vastenhouw N.L. Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos.Development. 2016; 143: 540-546Crossref PubMed Scopus (3) Google Scholar, Trcek et al., 2015Trcek T. Grosch M. York A. Shroff H. Lionnet T. Lehmann R. Drosophila germ granules are structured and contain homotypic mRNA clusters.Nat. Commun. 2015; 6: 7962Crossref PubMed Google Scholar). Second, validated workflows to analyze RNA localization and abundance will be of crucial importance and will ideally include information about the micro-environment of each cell, since it impacts gene expression (Battich et al., 2015Battich N. Stoeger T. Pelkmans L. Control of transcript variability in single mammalian cells.Cell. 2015; 163: 1596-1610Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). A common strategy to visualize single molecules in live cells uses repeated tags (Figure 3A). These tags bind multiple molecules of a fluorescent detector, thereby revealing single molecules of the target as diffraction-limited spots (see Figures 1C and 1E for examples). Single DNA loci were first visualized using a LacI-GFP fusion and a repetition of 256 lacO sites (Robinett et al., 1996Robinett C.C. Straight A. Li G. Willhelm C. Sudlow G. Murray A. Belmont A.S. In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.J. Cell Biol. 1996; 135: 1685-1700Crossref PubMed Scopus (515) Google Scholar). Likewise, RNA molecules were visualized using the coat protein of bacteriophage MS2 (Bertrand et al., 1998Bertrand E. Chartrand P. Schaefer M. Shenoy S.M. Singer R.H. Long R.M. Localization of ASH1 mRNA particles in living yeast.Mol. Cell. 1998; 2: 437-445Abstract Full Text Full Text PDF PubMed Google Scholar; Figure 3A). This protein binds an RNA stem-loop of 19 nucleotides and repetition of 24 such stem-loops in a reporter RNA allows its detection with single-molecule sensitivity (Fusco et al., 2003Fusco D. Accornero N. Lavoie B. Shenoy S.M. Blanchard J.M. Singer R.H. Bertrand E. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells.Curr. Biol. 2003; 13: 161-167Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). More recently, a similar technique (SunTag) was developed for proteins (Tanenbaum et al., 2014Tanenbaum M.E. Gilbert L.A. Qi L.S. Weissman J.S. Vale R.D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging.Cell. 2014; 159: 635-646Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar; Figure 3B). Here, 12 to 24 repetitions of an epitope are added to the protein of interest and are detected with a monochain antibody fused to GFP. There are now a number of tag variants that enable multicolor detection of multiple RNA species (reviewed in Tutucci et al., 2018aTutucci E. Livingston N.M. Singer R.H. Wu B. Imaging mRNA in vivo, from birth to death.Annu. Rev. Biophys. 2018; 47: 85-106Crossref PubMed Scopus (20) Google Scholar). In particular, orthogonal RNA labeling can be done with the coat protein of phage PP7 (PCP), as well as the human U1A protein, and the λN and BlgG bacterial anti-terminators. In all these approaches, unbound molecules of detector result in background signal, but this can be reduced by targeting them to a different cellular compartment (Bertrand et al., 1998Bertrand E. Chartrand P. Schaefer M. Shenoy S.M. Singer R.H. Long R.M. Localization of ASH1 mRNA particles in living yeast.Mol. Cell. 1998; 2: 437-445Abstract Full Text Full Text PDF PubMed Google Scholar) or by fine-tuning detector expression (Fusco et al., 2003Fusco D. Accornero N. Lavoie B. Shenoy S.M. Blanchard J.M. Singer R.H. Bertrand E. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells.Curr. Biol. 2003; 13: 161-167Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, Wu et al., 2012Wu B. Chao J.A. Singer R.H. Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells.Biophys. J. 2012; 102: 2936-2944Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). To ensure that the investigated process is not altered by the labeling method, untagged and tagged RNA can be compared by smFISH, with and without detector expression. MCP, PCP, and the SunTag have all been optimized for solubility, affinity, and specificity. An advantage of the MCP system is that this RNA-protein interaction has been intensively studied, enabling its fine-tuning for particular applications. Lower affinity variants of the system allow artifact-free studies of RNA metabolism (Tantale et al., 2016Tantale K. Mueller F. Kozulic-Pirher A. Lesne A. Victor J.M. Robert M.C. Capozi S. Chouaib R. Bäcker V. Mateos-Langerak J. et al.A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting.Nat. Commun. 2016; 7: 12248Crossref PubMed Google Scholar; see a detailed study in Tutucci et al., 2018bTutucci E. Vera M. Biswas J. Garcia J. Parker R. Singer R.H. An i" @default.
• W2886822966 created "2018-08-22" @default.
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• W2886822966 date "2018-08-01" @default.
• W2886822966 modified "2023-10-13" @default.
• W2886822966 title "A Growing Toolbox to Image Gene Expression in Single Cells: Sensitive Approaches for Demanding Challenges" @default.
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